Infection with certain human papillomavirus (HPV) types such as HPV16 and HPV18 is responsible for more than 5% of all human cancers worldwide. Despite the introduction of a prophylactic vaccine in 2006, HPV will continue to cause significant morbidity and mortality in the human population due to the long latency period between infection and disease, limited vaccine coverage, and type-restricted neutralizing immune responses. Therefore, further studies into the understanding of the biology of this virus group are warranted. We have been interested in how HPV16 establishes infection using in vitro infection models. In recent years, we have gained a good understanding of cell surface and subsequent events like internalization and trafficking to the perinuclear space of host cells. The trans-golgi network (TGN) serves as intermediate target organelle of the cytoplasmic viral transport and the final target are the PML nuclear bodies (NB), subnuclear structures targeted by many DNA viruses. However, little is known about the final steps of entry, including the escape from the TGN compartment and trafficking to the nucleus. It has previously been established that nuclear envelope break down is required for viral genome to enter the nucleus. We recently found that viral genome dissociates from TGN in prophase and associates with microtubules (MT) to accumulate at the microtubule-organizing center, implying minus-end directed transport along MT. At later stages of mitosis (metaphase), viral genome moves away from the mitotic spindle poles towards chromosomes suggesting a switch to plus-end directed transport. At this stage, viral genome is still found in a membrane-bound vesicular compartment based on its inaccessibility to small molecule dyes and nucleases. We also find that viral genome is deposited onto mitotic chromosomes in the vicinity of kinetochores. Viral genome becomes accessible to dyes after nuclear envelope reformation in interphase cells suggesting that viral genome egresses from the endocytic compartment only after mitosis has been completed and the nuclei have reformed. Release from the transport vesicle is preceded by PML protein recruitment. In cells deficient for PML protein, the core component of PML NB, viral genome is lost and therefore transcriptionally inactive. Non-enveloped DNA viruses need to navigate many hurdles during infectious entry into host cells including the passage over cellular membranes and the escape from innate immune sensors. Our preliminary data suggest that HPV achieves the escape from cytosolic sensors by hiding inside vesicles until the virus has entered the nucleus during mitosis and until the nuclear envelope has reformed. Taken together, our findings make it plausible that HPV utilizes transport vesicles that bud out of the Golgi compartment at the onset of mitosis for MT-mediated transport towards mitotic chromosomes. Our data point to a switch in the directionality of MT- mediated transport from minus end- to plus end-directed during mitosis, which will be studied in Aim 1. The simplicity of the HPV system may help unravel this switch in directionality, which is incompletely understood in most viral infections. The identification of a mutant virus defective for plus end-directed transport will aid us in our mechanistic studies. Aim 2 is dedicated to understanding the role of PML NB components in establishment of HPV16 infection. Many, if not all, DNA viruses target PML NB and reorganize them. However, they also reassemble a modified form of PML NB for efficient establishment of infection. In contrast, HPV requires PML protein for establishment of infection. Our observations that PML NB protects incoming HPV genome from degradation may provide an explanation for these findings. Another PML NB-resident protein, Sp100, has been shown to restrict HPV18 transcription, suggesting that PML NBs play a prominent role in establishment of HPV infection. We have now developed a novel cell culture model that allows efficient infection of primary keratinocytes for investigating the role of PML NB components in a relevant cell culture model. Overall, our proposed studies will fill a huge gap in the understanding of MT-mediated transport during late HPV intracellular trafficking and will help us unravel the role of PML NB during the immediate early events of the HPV life cycle essential for establishing infection, which may also shed light onto the as of yet incompletely understood functions of these subnuclear structure in viral infection and cell homeostasis.